John P. Hays

An RNA transcription‐based amplification technique (NASBA) for the detection of viable Salmonella enterica

S.A. SIMPKINS, A.B. CHAN, J. HAYS, B. PÖPPING & N. COOK.2000.Possession of mRNA is indicative of cell viability. RTPCR is not appropriate for mRNA detection as it cannot unambiguously detect mRNA in a DNA background. The alternative amplification technique, NASBA, avoids the disadvantages of RTPCR. We have devised a method for detection of viable Salmonella enterica. This involves NASBA amplification of mRNA transcribed from the dnaK gene. Amplification of mRNA extracted from viable and heat‐killed cells from the same population produced consistent and highly significant (P > 0·01) differences between the respective signals. The signal obtained from viable cells was completely eradicated by RNase treatment, while PCR amplification of treated and untreated samples was unaffected, indicating that NASBA was unaffected by background DNA.

Genome analysis of Moraxella catarrhalis strain RH4, a human respiratory tract pathogen

Moraxella catarrhalis is an emerging human-restricted respiratory tract pathogen that is a common cause of childhood otitis media and exacerbations of chronic obstructive pulmonary disease in adults. Here, we report the first completely assembled and annotated genome sequence of an isolate of M. catarrhalis, strain RH4, which originally was isolated from blood of an infected patient. The RH4 genome consists of 1,863,286 nucleotides that form 1,886 protein-encoding genes. Comparison of the RH4 genome to the ATCC 43617 contigs demonstrated that the gene content of both strains is highly conserved. In silico phylogenetic analyses based on both 16S rRNA and multilocus sequence typing revealed that RH4 belongs to the seroresistant lineage. We were able to identify almost the entire repertoire of known M. catarrhalis virulence factors and mapped the members of the biosynthetic pathways for lipooligosaccharide, peptidoglycan, and type IV pili. Reconstruction of the central metabolic pathways suggested that RH4 relies on fatty acid and acetate metabolism, as the genes encoding the enzymes required for the glyoxylate pathway, the tricarboxylic acid cycle, the gluconeogenic pathway, the nonoxidative branch of the pentose phosphate pathway, the beta-oxidation pathway of fatty acids, and acetate metabolism were present. Moreover, pathways important for survival under challenging in vivo conditions, such as the iron-acquisition pathways, nitrogen metabolism, and oxidative stress responses, were identified. Finally, we showed by microarray expression profiling that approximately 88% of the predicted coding sequences are transcribed under in vitro conditions. Overall, these results provide a foundation for future research into the mechanisms of M. catarrhalis pathogenesis and vaccine development.

Molecular Aspects of Moraxella catarrhalis Pathogenesis

SUMMARY In recent years, Moraxella catarrhalis has established its position as an important human mucosal pathogen, no longer being regarded as just a commensal bacterium. Further, current research in the field has led to a better understanding of the molecular mechanisms involved in M. catarrhalis pathogenesis, including mechanisms associated with cellular adherence, target cell invasion, modulation of the hosts immune response, and metabolism. Additionally, in order to be successful in the host, M. catarrhalis has to be able to interact and compete with the commensal flora and overcome stressful environmental conditions, such as nutrient limitation. In this review, we provide a timely overview of the current understanding of the molecular mechanisms associated with M. catarrhalis virulence and pathogenesis.

Comparative analysis and supragenome modeling of twelve Moraxella catarrhalis clinical isolates

BackgroundM. catarrhalis is a gram-negative, gamma-proteobacterium and an opportunistic human pathogen associated with otitis media (OM) and exacerbations of chronic obstructive pulmonary disease (COPD). With direct and indirect costs for treating these conditions annually exceeding

Antimicrobial resistance trends in blood culture positive Salmonella Typhi isolates from Pondicherry, India, 2005–2009

33 billion in the United States alone, and nearly ubiquitous resistance to beta-lactam antibiotics among M. catarrhalis clinical isolates, a greater understanding of this pathogens genome and its variability among isolates is needed.ResultsThe genomic sequences of ten geographically and phenotypically diverse clinical isolates of M. catarrhalis were determined and analyzed together with two publicly available genomes. These twelve genomes were subjected to detailed comparative and predictive analyses aimed at characterizing the supragenome and understanding the metabolic and pathogenic potential of this species. A total of 2383 gene clusters were identified, of which 1755 are core with the remaining 628 clusters unevenly distributed among the twelve isolates. These findings are consistent with the distributed genome hypothesis (DGH), which posits that the species genome possesses a far greater number of genes than any single isolate. Multiple and pair-wise whole genome alignments highlight limited chromosomal re-arrangement.ConclusionsM. catarrhalis gene content and chromosomal organization data, although supportive of the DGH, show modest overall genic diversity. These findings are in stark contrast with the reported heterogeneity of the species as a whole, as wells as to other bacterial pathogens mediating OM and COPD, providing important insight into M. catarrhalis pathogenesis that will aid in the development of novel therapeutic regimens.

Colonization of healthy children by Moraxella catarrhalis is characterized by genotype heterogeneity, virulence gene diversity and co-colonization with Haemophilus influenzae.

Typhoid fever is caused by Salmonella enterica serovar Typhi, a major public health concern in developing countries. Recently, there has been an upsurge in the occurrence of bacterial isolates that are resistant to ciprofloxacin, and the emergence of broad spectrum β-lactamases in typhoidal salmonellae constitutes a new challenge for the clinician. A total of 337 blood culture isolates of S. Typhi, isolated from Pondicherry, India, between January 2005 and December 2009, were investigated using phenotypic, molecular and serological methods. Of the 337 isolates, 74 (22%) were found to be multidrug resistant (MDR) and 264 (78%) nalidixic acid resistant (NAR). Isolates with reduced susceptibility to ciprofloxacin possessed single mutations in the gyrA gene. A high rate of resistance (8%) was found to ciprofloxacin. All isolates with a ciprofloxacin MIC ≥ 4 mg/L possessed both double mutations in the QRDR of the gyrA gene and a single mutation in the parC gene. Active efflux pump mechanisms were also found to be involved in ciprofloxacin resistance. Finally, a large number of PFGE patterns (non-clonal genotypes) were observed among the S. Typhi isolates. In conclusion, a high rate of ciprofloxacin resistance was observed in comparison to other endemic areas in blood culture isolates of S. Typhi from Pondicherry, India, with steadily increasing NAR but decreasing MDR isolations over the study period. This is most likely to be due to an increased use of ciprofloxacin as a first-line drug of choice over more traditional antimicrobial agents for the treatment of typhoid fever.

Analysis of VanA vancomycin-resistant Enterococcus faecium isolates from Saudi Arabian hospitals reveals the presence of clonal cluster 17 and two new Tn1546 lineage types

The colonization dynamics of Moraxella catarrhalis were studied in a population comprising 1079 healthy children living in Rotterdam, The Netherlands (the Generation R Focus cohort). A total of 2751 nasal swabs were obtained during four clinic visits timed to take place at 1.5, 6, 14 and 24 months of age, yielding a total of 709 M. catarrhalis and 621 Haemophilus influenzae isolates. Between January 2004 and December 2006, approximate but regular 6-monthly cycles of colonization were observed, with peak colonization incidences occurring in the autumn/winter for M. catarrhalis, and winter/spring for H. influenzae. Co-colonization was significantly more likely than single-species colonization with either M. catarrhalis or H. influenzae, with genotypic analysis revealing no clonality for co-colonizing or single colonizers of either bacterial species. This finding is especially relevant considering the recent discovery of the importance of H. influenzae-M. catarrhalis quorum sensing in biofilm formation and host clearance. Bacterial genotype heterogeneity was maintained over the 3-year period of the study, even within this relatively localized geographical region, and there was no association of genotypes with either season or year of isolation. Furthermore, chronological and genotypic diversity in three immunologically important M. catarrhalis virulence genes (uspA1, uspA2 and hag/mid) was also observed. This study indicates that genotypic variation is a key factor contributing to the success of M. catarrhalis colonization of healthy children in the first years of life. Furthermore, variation in immunologically relevant virulence genes within colonizing populations, and even within genotypically identical M. catarrhalis isolates, may be a result of immune evasion by this pathogen. Finally, the factors facilitating M. catarrhalis and H. influenzae co-colonization need to be further investigated.

Age-related genotypic and phenotypic differences in Moraxella catarrhalis isolates from children and adults presenting with respiratory disease in 2001-2002.

OBJECTIVES The aim of this study was to characterize 34 vancomycin-resistant VanA Enterococcus faecium isolates obtained from two hospitals in Saudi Arabia and to assess Tn1546 variation within these isolates. METHODS PFGE and multilocus sequence typing (MLST) genotypes, antibiotic susceptibility patterns, the presence of enterococcal surface protein (esp) and hyaluronidase (hyl) genes and conjugation frequencies were determined. In addition, Tn1546 elements were characterized. RESULTS PFGE and MLST analysis revealed the presence of 31 and 6 different genotypes, respectively. Further, three new ST types were discovered. Ninety-seven percent (33/34) of the isolates were associated with clonal complex 17 (CC17), with all isolates but one being resistant to ampicillin and all isolates being susceptible to linezolid. The esp and hyl genes were found in 44% (15/34) and 53% (18/34) of the isolates, respectively. Tn1546 analysis revealed that the isolates belonged to five different groups, including two new lineages. The IS-element insertions described did not abolish the transfer of VanA resistance. CONCLUSIONS VanA vancomycin-resistant E. faecium isolates obtained from Saudi Arabian hospitals include CC17 MLST types, a clonal cluster associated with E. faecium nosocomial infection worldwide. Novel E. faecium MLST types are circulating in Saudi Arabia, as well as novel Tn1546 types. It seems likely that CC17 E. faecium isolates may be distributed throughout the Middle East as well as Europe, America, Africa and Australia.

bro β-lactamase and antibiotic resistances in a global cross-sectional study of Moraxella catarrhalis from children and adults

Moraxella catarrhalis is generally associated with upper respiratory tract infections in children and lower respiratory tract infections in adults. However, little is known regarding the population biology of isolates infecting these two age groups. To address this, a population-screening strategy was employed to investigate 195 worldwide M. catarrhalis isolates cultured from children (<5 years of age) and adults (>20 years of age) presenting with respiratory disease in the years 2001-2002. Parameters compared included: genotype analysis; autoagglutination/biofilm-forming ability; serum resistance; uspA1, uspA2, uspA2H, hag and mcaP incidence; copB/LOS/ompCD/16S rRNA types; and UspA1/Hag expression. A significant difference in biofilm formation (P=0.002), but not in autoagglutination or serum resistance, was observed, as well as significant differences in the incidence of uspA2- and uspA2H-positive isolates, and the distribution of lipooligosaccharide (LOS) types (P<0.0001 and P=0.01, respectively). Further, a significant decrease in the incidence of Hag expression (for isolates possessing the hag gene) was observed in adult isolates (P=0.001). Both uspA2H and LOS type B were associated with 16S rRNA type 1 isolates only, and two surrogate markers (copB and ompCD PCR RFLP types) for the two major M. catarrhalis 16S rRNA genetic lineages were identified. In conclusion, there are significant differences in phenotype and gene incidence between M. catarrhalis isolates from children and adults presenting with respiratory disease, possibly as a result of immune evasion in the adult age group. Our results should also be useful in the choice of effective vaccine candidates against M. catarrhalis.

Highly specific protease-based approach for detection of porphyromonas gingivalis in diagnosis of periodontitis.

OBJECTIVES To compare and contrast the geographic and demographic distribution of bro beta-lactamase and antibiotic MIC(50/90) for 1440 global Moraxella catarrhalis isolates obtained from children and adults between 2001 and 2002. METHODS One thousand four hundred and forty M. catarrhalis isolates originating from seven world regions were investigated. The isolates were recovered from 411 children <5 years of age and 1029 adults >20 years of age. PCR-restriction fragment length polymorphism (RFLP) was performed to determine bro prevalence and to distinguish between bro types. MIC values of 12 different antibiotics were determined using the CLSI (formerly NCCLS) broth microdilution method. RESULTS Of the 1440 isolates, 1313 (91%) possessed the bro-1 gene and 64 (4%) possessed the bro-2 gene. Additionally, the prevalence of bro positivity between the child and adult age groups was significantly different (P < 0.0001), though bro-1 and bro-2 prevalences within age groups were not significantly different. Consistently higher beta-lactam MICs were observed for M. catarrhalis isolates originating in the Far East. Significant correlations in MICs were observed for several antibiotic combinations, including all five beta-lactams with each other, and among the two quinolones. CONCLUSIONS The worldwide prevalence of bro gene carriage in clinical isolates of M. catarrhalis is now approaching 95%, with children significantly more likely to harbour bro-positive isolates than adults. Further, statistically significant differences in the distribution of beta-lactam MICs were observed between different world regions, particularly with respect to the Far East.